Azidoproline containing helices: Stabilization of the polyproline II structure by a functionalizable group

Article abstract of DOI:10.1021/ja067148o

The conformation and functionalizability of 4-azidoproline containing polyprolines was studied. The conformational analyses demonstrate that the PPII helix is stabilized by (4R)Azp and destabilized by (4S)Azp. Functionalization of Azp containing polyprolines was accomplished efficiently in a differential fashion by "click chemistry", rendering Azp containing polyprolines attractive molecular scaffolds. Copyright

Full text of DOI:10.1021/ja067148o

Published on Web 12/29/2006
Azidoproline Containing Helices: Stabilization of the Polyproline II Structure
by a Functionalizable Group
Michael K u¨ min, Louis-Sebastian Sonntag, and Helma Wennemers*
Department of Chemistry, UniVersity of Basel, St. Johanns-Ring 19, CH-4056 Basel, Switzerland
Received October 5, 2006; E-mail: helma.wennemers@unibas.ch
The polyproline II (PPII) helix is a common secondary structure
within natural proteins and plays important roles in many biological
1
processes. The development of molecular probes that allow for
influencing the conformational stability of the PPII structure is
1-3
therefore an important goal.
Likewise, conformationally well-
defined molecular scaffolds that allow for facile functionalization
have become increasingly important for applications ranging from
the generation of functional materials to the development of cell-
penetrating compounds, antibiotics, and inhibitors for specific
protein-protein interactions.4 Here we demonstrate that 4-azi-
doprolines can be used both as conformation directing elements of
the PPII structure and as functionalizable sites for the development
of proline-based molecular scaffolds.
,5
Figure 2. CD-spectra of Ac-[(4R)Azp]9-OH 2R (left) and Ac-[(4S)Azp]9-
OH 2S (right) in aqueous phosphate buffer (10 mM, pH 7.2) (red), 25%
vol/vol n-PrOH in buffer (dark purple), 50% n-PrOH (yellow), 75% n-PrOH
Short proline oligomers adopt the PPII conformation in aqueous
solution, and changing to a less polar solvent, like aliphatic alcohols,
can induce a different secondary structure, the polyproline I (PPI)
helix. The PPII conformation is a left-handed helix with all amide
bonds in s-trans conformations and every third residue stacked on
top of each other in a lateral distance of 9.4 Å.6 The PPI helix is
right-handed and more compact (helical pitch of 5.6 Å per turn)
with all amide bonds in s-cis conformations and 3.3 proline residues
per turn.6 The characteristic well-defined conformation of oligo-
prolines in water, combined with the possibility of switching from
(
aqua), 85% n-PrOH (light purple), 90% n-PrOH (black), 95% n-PrOH
(green), and n-PrOH (blue). Spectra were recorded at concentrations of
-
4
6
× 10 M per residue at 25 °C.
phosphate buffer (10 mM, pH 7.2), n-PrOH, and mixtures thereof
by CD-spectroscopy (Figure 2).
,7
The CD-spectrum of 2R in phosphate buffer is typical for a PPII
6
helix (maximum at 225, minimum at 207 nm) and hardly any
,7
conformational change occurs up to a content of 90% vol/vol of
n-PrOH in buffer. Only in pure n-PrOH is the CD spectrum of 2R
indicative of a PPI helix (minimum at 230 nm, maximum at 212
nm). In contrast, the conformation of 2S changes drastically toward
PPI when n-PrOH is present in aqueous buffer (Figure 2). Analysis
7
one conformation to another, render oligoprolines interesting as
molecular scaffolds, if attachment sites can be introduced that allow
for site-specific placement of functionalized side chains.
Previous studies on (4R)- or (4S)-azidoprolines (Azp) had
revealed that their conformation is governed by an “azido gauche
9
of the unmodified 9-mer of proline, Ac-[Pro] -OH, revealed that
its conformational stability is in between those of 2R and 2S (see
Supporting Information). These results demonstrate that (4R)Azp
effect” which leads to significantly different s-trans/s-cis isomer
8
ratios around their amide bonds (Figure 1). For Ac-(4R)Azp-OCH
3
9
stabilizes whereas (4S)Azp destabilizes the PPII conformation. The
1
2
4
R and Ac-(4S)Azp-OCH
.6:1, respectively, were observed in D
.6:1 for Ac-Pro-OCH . The higher s-trans:s-cis isomer ratio
3
1S s-trans/s-cis ratios of 6.1:1 and
data suggest that the stabilizing effect of (4R)Azp is due to an
2
O compared to a ratio of
enhancement of the n f π* interactions which have been proposed
2
3
to stabilize the PPII conformation. Comparable observations have
observed for the (4R)Azp derivative was rationalized on the basis
of a stabilizing n f π* interaction between the oxygen of the acetyl
group with the carbonyl group of the methylester of the s-trans
conformer of 1R.2
10
been made by Raines et al. with 4-fluoroprolines, suggesting that
the effect of an azide is comparable to that of a fluorine. In contrast
11
to fluorine, azido groups allow for further functionalization.
,8
To evaluate the functionalizability of Azp containing oligopro-
6
lines, we prepared oligoproline Ac-[Pro-(4R)Azp-Pro] -OH 3R with
Azp residues in every third position. 3R was used as a model
compound, designed to have all Azp residues stacked on top of
each other at one edge of the PPII helix (Figure 3A). Conformational
analyses of 3R in aqueous solution support this geometry. The CD-
spectrum in phosphate buffer is typical of a PPII conformation and
Figure 1. s-Trans/s-cis isomer ratios of 1R and 1S.
1
13
H and C NMR spectra show essentially one set of signals for
These results suggest that (4R)Azp and (4S)Azp could be used
to tune the stability of the PPII conformation. In addition, the azido
groups of Azp containing oligoprolines would provide versatile sites
for functionalization of the polyproline scaffold.
each, the Azp and the two flanking Pro residues. In n-PrOH, the
CD-spectrum of 3R is typical of a PPI-helix.
Functionalization of 18-mer 3R was accomplished using Huis-
gen’s 1,3-dipolar cycloaddition (“click reaction”).12 Reaction of the
To test the influence of (4R)Azp and (4S)Azp on the PPII
six azido groups with methyl propiolate, CuSO
4 2
‚5H O, and sodium
conformation, we synthesized the 9-mers Ac[(4R)Azp]
9
OH 2R and
ascorbate led to the desired hexatriazole 4R which was isolated by
Ac[(4S)Azp] OH 2S and studied their conformations in aqueous
9
simple extraction and precipitation in 40% yield (Figure 3B).
466
9
J. AM. CHEM. SOC. 2007, 129, 466-467
10.1021/ja067148o CCC: $37.00 © 2007 American Chemical Society

C O M M U N I C A T I O N S
polyprolines can be efficiently functionalized in a differential way
using “click chemistry”. Upon functionalization the well-defined
PPII conformation is retained, thereby allowing for the positioning
of functional groups at desired sites. In light of the increasing
importance of molecular scaffolds,4 the presented Azp-containing
oligoprolines might serve as interesting new leads.
,5
Acknowledgment. This work was supported by the company
BACHEM by an endowed professorhip to H.W. and the Swiss
National Science Foundation as well as Ph.D. fellowships from
Novartis to M.K. and the Fonds der Chemischen Industrie to L.S.S.
We thank Prof. C. Ochsenfeld and S. Schweizer for generating the
molecular models, Prof. T. Kiefhaber for the use of his CD-
spectrometer, and him as well as Prof. J. Engel for useful
discussions.
Supporting Information Available: Experimental details on the
syntheses and analyses of compounds 2S and 2R-6R. This material
is available free of charge via the Internet at http://pubs.acs.org.
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